Honda Masayoshi, Razzaghi Mortezaali, Gaur Paras, Malacaria Eva, Marozzi Giorgia, Di Biagi Ludovica, Aiello Francesca Antonella, Paintsil Emeleeta A, Stanfield Andrew J, Deppe Bailey J, Gakhar Lokesh, Schnicker Nicholas J, Spies M Ashley, Pichierri Pietro, Spies Maria
Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA.
Mechanisms, Biomarkers and Models section, Department of Environment and Health, Istituto Superiore di Sanità, Rome, Italy.
Nature. 2025 May;641(8062):512-519. doi: 10.1038/s41586-025-08753-1. Epub 2025 Apr 2.
Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability, including protection of stalled DNA replication forks from excessive degradation. In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress, protecting them from reversal by SMARCAL1 motor. The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses, we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the single-stranded DNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1.
人类RAD52是一种多功能DNA修复蛋白，参与多种支持基因组稳定性的细胞事件，包括保护停滞的DNA复制叉不被过度降解。在其守门人角色中，RAD52在复制应激期间结合并稳定停滞的复制叉，保护它们不被SMARCAL1马达逆转。RAD52介导的叉保护的结构和分子机制仍然难以捉摸。在这里，我们使用P1核酸酶敏感性、生化和单分子分析表明，RAD52通过其链交换活性动态重塑复制叉。叉处单链DNA结合蛋白RPA的存在调节链交换的动力学，而不影响反应结果。质量光度法和单颗粒冷冻电子显微镜显示，复制叉促进了一种独特的核蛋白结构，该结构包含两个十一聚体RAD52环的头对头排列，其带正电荷的表面延伸，可容纳复制叉的所有三个臂。我们认为，该表面的形成和连续性对于链交换反应以及与SMARCAL1的竞争很重要。
